Epstein-Barr Virus (EBV) is an oncogenic human herpesvirus predominantly infecting B-lymphocytes that is etiologically associated with Burkitt's lymphoma, AIDS-related immunoblastic lymphomas, post- transplant lymphoproliferative disease, nasopharyngeal carcinoma, and gastric carcinoma. Very few viral genes are expressed in these malignancies and diseases, and infectious virus is almost never released. Therefore, this type of EBV infection is called latency. There are three patterns of viral gene expression observed during latency, termed Type I, Type II and Type III. A nuclear EBV protein called the Epstein-Barr nuclear antigen 1 (EBNA1) is expressed during all three types of latency, but its function differs between latency types. In Type II and Type III latency, EBNA1 activates transcription of other viral genes required for cell proliferation, such as LMP1 and EBNA2. Type II latency is usually observed in EBV-associated carcinomas, whereas Type III latency is observed in AIDS-related immunoblastic lymphomas, post-transplant lymphoproliferative disease, and when EBV immortalizes naive B-cells in vitro. EBNA1 does not transactivate viral genes during Type I latency, which is observed in malignancies such as Burkitt's lymphomas. However, in such cells, EBNA1 has been reported to transactivate the expression of cellular genes. In addition, expression of a dominant-negative EBNA1 that cannot activate transcription severely reduces the aggressive proliferation of EBV-positive Burkitt's lymphoma cells, and induces apoptosis in the majority of cells. Surprisingly, in contrast to these results, over-expression of EBNA1 inhibits the growth of EBV-negative Burkitt's lymphoma cells. Therefore, the ability of EBNA1 to transactivate has contrasting effects on the growth and survival of EBV-positive and EBV-negative Burkitt's lymphomas. A study published recently demonstrates that EBNA1 induces reactive oxygen species (ROS) when expressed in Burkitt's lymphomas, and thereby negatively affects genomic stability. EBNA1 generates ROS by increasing the expression of cytochrome b-245 heavy chain (NOX2). Recent studies on the transactivation mechanism of EBNA1 have shown that EBNA1 needs three domains to transactivate: a) two domains that bind AT-rich DNA, and are termed AT-hooks;and b) a domain called unique region 1 (UR1) that contains an essential conserved cys-x-x-cys motif that permits EBNA1 to dimerize by coordinating zinc. The two cysteines in UR1 are regulated by oxidative stress (redox), such that under oxidative conditions, EBNA1 does not transactivate viral genes. These conditions also interfere with the proliferation of B-cells transformed by EBV in vitro. I propose investigating the functions and domains of EBNA1 required for proliferation of EBV-immortalized lymphoblastoid cell lines, and EBNA1 functions necessary for the aggressive proliferation of malignant Burkitt's lymphoma cells. My studies will identify new therapeutic targets against diseases and malignancies caused by latent EBV infections. PUBLIC HEALTH RELEVANCE: The NCI SEER Cancer Statistics Review indicates that an estimated 75000 men and women will be diagnosed with, and 20,000 individuals will die from lymphoma this year in the US. Infection by Epstein-Barr virus results in 30-50% of B-cell lymphomas. This goal of my research is to yield therapeutics that decrease those numbers, and therefore augment human health and welfare.